Stimulation of homologous recombination in eukaryotic organisms or cells by recombination promoting enzymes

ABSTRACT

The present invention relates to a novel process for the production of transgenic organisms or transgenic cells, to transgenic orgaisms or transgenic cells obtainable by the process of the present invention, to the use of vectors comprising DNA encoding a recombination promoting enzymes for curing impairments caused by environmental influences in plants or plant cells and for gene therapy in mammals or mammalian cells, and to novel vectors.

This application is the national phase under 35 U.S.C. § 371 of priorPCT International Application No. PCT/EP96/03824 which has anInternational filing date of Aug. 30, 1996 which designated the UnitedStates of America, the entire contents of which are hereby incorporatedby reference.

FIELD OF THE INVENTION

The present invention relates to a novel process for the production oftransgenic organisms or transgenic cells, to transgenic organisms ortransgenic cells obtainable by the process of the present invention, tothe use of vectors comprising DNA encoding a recombination promotingenzymes for curing impairments caused by environmental influences inplants or plant cells and for gene therapy in mammals or mammaliancells, and to novel vectors.

The process of homologous recombination requires search for homology,recognition of sequence similarity, and strand exchange between two DNAmolecules. In bacteria, these different steps are mediated by a singleprotein, the RecA protein (for review see:Roca and Cox, 1990), whichplays a central role in the recombination pathway of E. coli. However,additional proteins are needed to initiate recombination and to resolvethe intermediates created by RecA. Recombination is initiated by thegeneration of single-stranded DNA (ssDNA) and DNA ends in E. coli andpresumably in all organisms. In E. coli, the combined action of theproducts of the recB, recC, and recD genes initiates a majorrecombination pathway (for review see: Dunderdale and West, 1994). ssDNAis recognised by RecA protein and double-stranded DNA (dsDNA) isactively searched for. Exchange of complementary strands leads to theformation of recombination intermediates (Holliday structures). Theintermediates can be resolved by different pathways; the major oneinvolves the action of the RuvA, RuvB, and RuvC proteins. All of therecombination proteins have to work in concert to complete recombinationsuccessfully. Proteins remarkably similar to RecA have been found in anumber of eukaryotic cells such as budding yeast, fission yeast, humans,mice, chicken, and plants (Terasawa et al., 1995; for review see:Kowalczykowski and Eggleston, 1994). The best characterised ones are theDmc1 and Rad51 proteins from Saccharomyces cerevisiae. In both cases thecorresponding genes are essential for recombination and the proteinsshow considerable sequence homology to RecA. A comparison of the primarysequences of Dmc1 and several bacterial RecA proteins suggests thatthese proteins evolved from a single progenitor before the separation ofprokaryotes and eukaryotes. In addition, Rad51 was shown to bestructurally very similar to RecA. Rad51 forms DNA/protein filaments,strikingly similar in tertiary structure to those formed with RecA(Ogawa et al., 1993). While previous studies failed to showATP-dependent homologous pairing and strand-exchange mediated by Rad51(Shinohara, et al., 1992; Ogawa et al., 1993), more recent experimentshave demonstrated these activities (Sung, 1994). Rad51 interacts withother proteins, e.g. Rad52 and Dmc1, so Rad51 may be part of a complexinvolved in recombination.

However, the complexity of these proteins strongly argues against theirbeing simply a homologue as equivalent to the E.coli RecA protein.Accordingly, different modes of biological activity may be expected.

Various reports have been published focusing on the activity of E. coliRecA protein in animal and in particular in mammalian cells. Thus, Kidoet. al, 1992 report on the introduction of functional bacterial RecAprotein which was fused to the nuclear location signal of SV40 largeT-antigen into mammalian cells. However, no functional studies of theintroduced protein were carried out. WO 93/22443 deals with thetargeting of exogenous polynucleotide sequences coated on E. coli RecAprotein to chromosomal DNA of mammalian cells. This document shows thatRecA protein coated oligonucleotides can efficently be targeted tocorrect chromosomal positions, RecA can stimulate extrachromosomalrecombination, and RecA short DNA complexes can be used for genetargeting in mammalian cells. However, the authors failed to showstimulation of homologous recombination in living cells or an entireorganism. Spivak et. al (1991) report the increased survival of HeLacells upon treatment with RecA protein containing liposomes afterirradiation. However, RecA stimulated survival was only marginal.Cerruti et al. report on the recombinatorial activity of E.coli RecAprotein in plastids which, however, had no effect on DNA repair or cellsurvival, probably due to the fact that plastids have an ownrecombination promoting enzyme which is homologous to E.coli RecA. Thus,so far successful experiments with the goal of targeting RecA protein toeukaryotic nuclei which yield a significantly high recombinatorialactivity to allow for the industrial applicability of such processeshave not been carried out. For example, as regards plant cells,introduction of RecA/DNA complexes, in analogy to WO93/22443, in plantcells by PEG-mediated transformation turned out to be extremelydifficult. These complexes exhibit an apparent toxicity and lead to celldeath of nearly the total protoplast population. Also, Kido et. al, loc.cit., had reported on the failure to introduce RecA protein into thenuclei of mammalian cells. Therefore, in view of the prior artinvestigations it was highly questionable as to whether a recombinationpromoting enzyme such as RecA could be functionally introduced into thenuclei of eukaryotic cells and, furthermore, whether such an introducedRecA protein would indeed be able to enter the cell nucleus and activelypromote recombination to an industrially applicable extent.

SUMMARY OF THE INVENTION

Thus, the technical problem underlying the present invention was toprovide a process for the production of a transgenic organism or atransgenic cell, said process making use of a recombination promotingenzyme. The solution to said technical problem is provided by theembodiments characterised in the claims. Accordingly, the presentinvention relates to a process for the production of a transgenicorganism or a transgenic cell comprising

(a) insertion

(aa) of a DNA into the genome of an organism or a cell, said DNAcomprising a DNA which

(aaa) confers to the transgenic organism or the transgenic cell one ormore desired characteristics; which

(aab) additionally encodes at least one selection marker expressible insaid organism or said cell; and which

(aac) optionally encodes a recombination promoting enzyme or anenzymatically active derivative or part thereof, wherein therecombination promoting enzyme or the enzymatically active part thereofconfers the or one of the desired characteristics; or, if (aac) does notapply,

(ab) of a recombination promoting enzyme or an enzymatically activederivative or part thereof in combination with said DNA (aa), into anorganism or a cell;

(b) selection of transgenic organisms or cells, which have taken up saidDNA or said DNA and said protein according to (a); and

(c) culturing of the desired transgenic organism or the desiredtransgenic cell in a suitable culture medium.

Thus, it is conceivable in accordance with the present invention thatthe recombination promoting enzyme confers the desired characteristic oris one of the desired characteristics. In the first instance, the methodof the invention may yield, for example, plants with a hyperrecombinantphenotype which might be of use in plant breeding, plants more tolerantto environmental influences, for example, caused by UV or ozone, orplants more tolerant to DNA damage. In the second case, therecombination promoting enzyme may be used to introduce by promotingrecombination a DNA sequence of interest into the genome of a cell or anorganism. These transgenics are expected to improve the frequency ofgene targeting and make this methodology applicable, for example, forplant breeding.

Further, the recombination promoting enzyme may be introduced into thecell or organism as encoded by a corresponding nucleotide sequencewhich, upon expression, yields said recombination promoting enzyme.Alternatively, the recombination promoting enzyme may be introduced intosaid cell or organism as such. In this case, the DNA to be insertedencodes a protein with the second or further desired characteristic. Thenucleotide sequence encoding the second or further desiredcharacteristics may then be introduced into the genome of said cell ororganism by the activity of the recombination promoting enzyme, which,naturally, has to be biologically active in said cell or organism. Forexample, the second or further characteristic may be an additionalprotein to be expressed in said cell or organism or, it may be a DNAsequence that, upon recombination into the genome of said organism orsaid cell, results in the disruption of a naturally occurring or atransgenic gene function. This approach also allows the expression ofmodified proteins without interference endogenous non-modified copiesand allows to circumvent inter-transformant variation. It is alsoexpected that problems arising from instable expression and genesilencing can be circumvented using gene targeting in plants.

For testing the efficacy of the process of the invention, a reproducibleand quantitative assay for mitomycin C resistance was developed on thebasis of the data and systems published by Lebel et al. (1993).Mitomycin C is known to intercalate in vivo into DNA leading tocross-linking of complementary strands (Borowy-Borowski et al., 1990).Cross-linking leads to inhibition of DNA synthesis in bacteria withoutconcomitant effect on RNA or protein synthesis (Iyer and Szybalski,1963). In Ustilago maydis and Saccharomyces cerevisiae, mitomycin C wasshown to stimulate homologous recombination without being mutagenic(Holliday, 1964). Similar observations were also made in differenthigher eukaryotic cells (Suzuki, 1965; Shaw and Cohen, 1964; Wang etal., 1988). The data indicate that mitomycin C efficiently blocks DNAreplication. The resultant daughter-strand blocks are thought to berepaired in many organisms by homologous recombination (sister-chromatidexchange) and excision repair. Lebel et al. (1993) showed that mitomycinC stimulates intrachromosomal recombination in plant cells, thuspointing to recombinational repair of mitomycin C lesions in plants.

In accordance with the present invention it was found that highmitomycin C concentrations kill untreated plant cells as arepresentative of eukaryotic cells efficiently, presumably because thecapacity of the endogenous repair/recombination system is exhausted.Thus it was further found for the wild-type that protoplast survivalunder mitomycin C treatment followed a dose-response curve similar tothose frequently seen with bacteria and yeast: a shoulder at low dosesand a semi-logarithmic decrease at higher doses (FIG. 7). Evaluation ofthe dose-response curves (FIG. 7) as described by Friedberg (1985)suggests that nt-RecA expression obtained in accordance with the processof the present invention provides the cells with the capacity to repairdamage caused by up to 50 μg/ml mitomycin C whereas the endogenousrepair mechanism in wild-type tobacco protoplasts can only repair damagecaused by up to 15 μ/ml of mitomycin C.

Thus, plant cells expressing nt-RecA exhibited a considerably higherresistance to this drug. This suggests that RecA can function in plantcells, interacting with or supplementing the endogenous plantrecombination machinery. Furthermore, RecA directly stimulatedintrachromosomal recombination in plants. On the basis of this data itmay be expected that the process of the present invention yields muchhigher recombination frequencies than any of the processes described bythe prior art.

Although the present invention has been illustrated only with regard toplant cells, the teachings disclosed herein apply as well to othereukaryotic cells such as mammalian cells. It is expected that thepresent invention allows, for the first time, for a recombinationefficiency in the nuclei of eukaryotic cells or organisms that leads toan industrially applicable process for the generation of such transgeniccells or organisms.

In a preferred embodiment of the process of the invention, saidtransgenic organism or transgenic cell is a plant or a plant cell.

In a most preferred process of the present invention said plant or plantcells is or is derived from Nicotiana tabacum or Arabidopsis thaliana.

A further preferred embodiment of the invention relates to a methodwherein said transgenic organism as transgenic cell is a mammal or amammalian cell, a fungus, a yeast or a bacterium.

In a further preferred process said desired characteristics arestimulation of homologous recombination, enhancement of gene targeting,stimulation of endogenous mechanisms for repair of DNA damage, thusleading to tolerance to various chemical and physical agents (ozone,UV). Additionally, said further desired characteristic may, for example,be an additional protein expressed, which alters the phenotype of thetransgenic organism or cell in a desired way such as the expression ofadditional surface markers or the expression of different/additionalmetabolic enzymes. Further, said characteristic may lead to thedisruption of a naturally occurring gene function in said organism orsaid cell.

In another preferred process said selection marker is Hyg^(R), Km^(R),PPT^(R), Mtx^(R) or Sul^(R).

The person skilled in the art is well familiar with these selectionmarkers, where Hyg^(R) stands for hygromycin resistance, Km^(R) standsfor Kanamycin resistance, PPT^(R) stands for phosphonotricin (BASTA)resistance, Mtx^(R) stands for methotrexate resistance and Sul^(R)stands for sulfonamide resistance. The person skilled in the art is,however, able to replace these preferred selection markers by any otherone suitable in the process of the invention.

In an additional preferred embodiment of the process of the invention,said recombination promoting enzyme is the E.coli RecA protein.

In a further preferred process of the present invention said derivativeof said recombination promoting enzyme is a fusion protein of the E.coliRecA protein and a nuclear targeting sequence.

The experimental data obtained in accordance with this preferredembodiment showed that nt-RecA was able to increase the UV resistance ofrecA E. coli. The chimeric protein was capable of binding to ssDNA andto catalyse strand-exchange in vitro about as efficiently as RecA.Interestingly, unlike RecA, nt-RecA showed a high ATPase activity in theabsence of ssDNA. This ssDNA-independent activity of nt-RecA wasstimulated by addition of ssDNA by the same incremental amount as RecAitself. It is presently not known whether these activities result fromtwo different proteins (nt-RecA and a RecA-like degradation product) inthe preparation, or is an intrinsic property of nt-RecA. It can also notbe totally excluded that ATPase activity of nt-RecA in the absence ofssDNA is due to trace amounts of contaminating ATPase or DNA, which mayhave escaped detection by gel electrophoresis and staining. However, itis likely that the nt-RecA fusion protein indeed has different ATPaseproperties. Since the level of ATPase of nt-RecA in the absence of ssDNAis somewhat higher than that of RecA in the presence of ssDNA one mightthink of nt-RecA as a modified RecA protein which is constitutivelyactivated in a manner which is reminiscent of the RecA441 and RecA730proteins (Witkin et al., 1982). ATPase activity of RecA seems to servetwo different functions: recycling of the enzyme and overcomingnonhomologous regions of DNA (For review see: Kowalczykowski andEggelston, 1994; Roca and Cox, 1990).

In a most preferred embodiment said nuclear targeting sequence is the TSV40 nuclear targeting sequence.

The SV40 nuclear targeting sequence is well known in the art and neednot be described here any further.

In a still further preferred embodiment of the present invention saidinsertion is mediated via PEG transformation, Agrobacteriumtransformation, electroporation, particle bombardment, liposome fusion,in planta transformation, calcium phosphate precipitation, or virusinfection.

The optimal process employed depends on the taxonomic origin of theorganism or cell to be transfected. The person skilled in the art iswell aware of which insertion method is best suited to this purpose.

The invention further relates to a transgenic organism or a transgeniccell obtainable by the process of the invention.

Additionally, the invention relates to a vector comprising a DNAencoding a nuclear targeting sequence operatively linked to a DNAencoding a recombination promoting enzyme or an enzymatically activepart thereof, at least one selection marker, and, optionally, at leastone further DNA encoding a desired characteristic, wherein the nucleartargeting sequence/recombination promoting enzyme fusion protein encodedby said vector has ATPase activity.

Surprisingly, it was found in accordance with the present invention thatthe nuclear targeting sequence/recombination promoting enzyme fusionprotein as exemplified by the fusion protein wherein the nucleartargeting sequence is derived from SV40 and the recombination promotingenzyme is the E.coli RecA protein confers a high ATPase activity. Saidactivity appears to serve two different functions: First, it appears torecycle the enzyme and secondly, it appears to overcome non-homologousregions of DNA.

The at least one selection marker comprised in said vector which iscapable of expressing the various DNA sequences comprised therein, havebeen already discussed herein above. The same holds true for the furtherDNAs encoding a desired characteristic which may also be comprised inthe vector of the invention.

In a preferred embodiment of said vector, said nuclear targetingsequence is the T SV40 nuclear targeting sequence. Additionally oralternatively, the recombination promoting enzyme is the E.coli RecAprotein.

In a most preferred embodiment said vector of the invention ispS/nt-RecA or pEV/nt-RecA. The construction of said vectors is amplydescribed in Example 1. Further details of said vectors are given inFIG. 1.

Further, the invention relates to the use of a vector of the inventionor a vector comprising a DNA which confers to a cell to be transformedor transfected therewith one or more desired characteristics; said DNAadditionally encoding at least one selection marker expressible in saidcell and further encoding a recombination promoting enzyme or anenzymatically active derivative or part thereof, wherein therecombination promoting enzyme or the enzymatically active part thereofconfers the or one of the desired characteristics, for curingimpairments caused by environmental influences in plants or plantscells. As regards the selection markers, recombination promoting enzymesand insertion processes, preferred embodiments thereof have beendescribed herein above.

In a preferred embodiment of said use, said impairments are caused bydamage to DNA, preferably by UV irradiation, ozone, SO₂, methylatingagents or mutagenic agents.

In a final preferred embodiment of the invention, the vector describedherein above is used for gene therapy in mammals or mammalian cells.Such methods for gene therapy are amply discussed in the art so that thetechnical details are known to or derivable without further ado by theperson skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: SCHEMATIC REPRESENTATION OF TRANSGENIC RECA GENES.

Coding sequences are indicated by solid bars. In recA transgenes, openboxes indicate sequences which were added to the recA coding region.Small boxes indicate sequences not coding for proteins. The nucleotidesequences and corresponding amino acid sequences which was added to makepS/nt-recA and pEV/nt-recA are indicated. Promoters are shown as openarrows, polyadenylation signals as open boxes. Abbreviations: HindIII,recognition sequence for HindIII endonuclease; P35S, CaMV 35S promoter;PAA, polyadenylation signal from CaMV; ori, E. coli origin ofreplication; Sul^(r), sulfonamide resistance gene, Amp^(r), ampicillinresistance gene; Br, T-DNA right border sequence; Bl, T-DNA left bordersequence; λP_(l), leftward promoter of phage lambda.

FIG. 2: COMPARISON OF ATPASE ACTIVITIES OF RECA AND NT-RECA.

RecA and nt-RecA proteins (100 pMol) were respectively incubated withradioactively labeled ATP in the presence or absence of ssDNA. The ATPturnover was determined and specific activities calculated.

FIG. 3: COMPARISON OF SINGLE-STRAND BINDING ACTIVITIES OF RECA ANDNT-RECA.

0, 20, 50, and 100 pMol of RecA and nt-RecA protein respectively, wereincubated with 400 pMol (nucleotides) of ssDNA in the presence of γSATP.Reaction mixtures were analysed by electrophoresis on 0.8% agarose gelsat 4° C. and the DNA stained with ethidium bromide. Where indicated(+deprot.), samples were deproteinised prior to electrophoresis. Marker:Phage lambda DNA digested with PstI endonuclease. The position of thesingle-stranded substrate DNA in the gel is indicated (ssDNA).

FIG. 4: COMPARISON OF STRAND-EXCHANGE ACTIVITIES OF RECA AND NT-RECA.

RecA and nt-RecA proteins were incubated as described by Menetski et al.(1990), with mixtures of linear double-stranded and circular ssDNA inthe presence of single-strand binding protein (SSB) and γSATP, for thetime interval indicated in the figure. The mixture was analysed byelectrophoresis on 0.8% agarose gel at room temperature and the DNAstained with ethidium bromide. Control reactions which contained no RecAor nt-RecA protein (-(nt)RecA), no ySATP (-ATP), no magnesium (-Mg), nosingle-strand binding protein (-SSB), or no single-strand bindingprotein and γSATP(-SSB -ATP) were included. Marker M1 was phage lambdaDNA digested with PstI endonuclease, marker M2 was a mixture of relaxed(open circle, oc), linearised (lin), supercoiled (closed coiled circle,ccc) double-stranded and circular single-stranded (ss) DNA. The positionof the open circle, the reaction product of the strand-exchange reactionvisible in the gel, is indicated by an arrow.

FIG. 5: TRANSGENIC PLANTS EXPRESS RECA AND NT-RECA, RESPECTIVELY.

Proteins separated by electrophoresis on 12% polyacrylamide gelscontaining SDS were blotted to nitrocellulose, and RecA antigen detectedwith monoclonal anti-RecA antibody ARM414 (Ikeda et al, 1990). Theposition of RecA purified from E. coli is indicated. Molecular weightswere deduced from a mixture of pre-stained proteins (BioRad) which werecoelectrophoresed.

FIG. 6: SUBCELLULAR LOCALISATION OF RECA AND NT-RECA IN TRANSGENICTOBACCO PLANTS.

Protoplasts were prepared from leaves and the proteins fixed byformaldehyde. Nuclei were visualised by DAPI staining (DAPI panels). Thesame protoplasts were stained with anti-RecA antibody ARM414 and aFITC-labelled second antibody to visualise RecA antigen (Anti-RecApanels). A: protoplasts from non-transgenic SR1 plants. B: protoplastsfrom G64/2 plants expressing RecA. C.: protoplasts of G63/19 plantsexpressing nt-RecA.

FIG. 7: RECA AND NT-RECA TRANSGENIC PROTOPLASTS ARE MORE RESISTANT TOTHE TOXIC EFFECT OF MITOMYCIN C.

Protoplasts obtained from SR1, G64/2, and G63/19 plants were regeneratedto microcalli in the presence or absence of mitomycin C. Survivalfrequencies are defined by the number of microcalli regenerated in thepresence of mitomycin C divided by the number of microcalli regeneratedin its absence. Mean values obtained from three different data sets areshown. The error bars represent standard deviations calculated for eachdata point. Data points were reproduced within ±25% for 0, 10, 25, and50 μ/ml mitomycin C in three additional, independent experiments.

FIG. 8: RECA AND NT-RECA EXPRESSION LEADS TO A STIMULATION OFINTRACHROMOSOMAL RECOMBINATION.

At least 5×10⁶ protoplasts, prepared from each of the 3 lines, wereregenerated in the presence of kanamycin in three independentexperiments. The frequency of intrachromosomal recombination wascalculated from the number of calli growing in the presence ofkanamycin, divided by the regeneration frequency obtained in eachexperiment. Mean values are shown and error bars represent standarddeviations.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1 EXPRESSION AND NUCLEARTARGETING OF RECA IN PLANTS

Nuclear proteins concentrate in the nucleus irrespective of their size.“Nuclear localisation signals”, short stretches of amino acids, arebelieved to be responsible for targeting of nuclear proteins (forreviews see Dingwall and Laskey, 1986; Silver, 1991). The size of the E.coli RecA monomer is close to the exclusion limit of eukaryotic nuclearpores; cytoplasmic ssDNA/RecA filaments would most probably be excluded.RecA protein is unlikely to contain nuclear localisation signals andtherefore might be excluded from the nucleus and hence from its target.In order to cover this eventuality RecA protein was expressed in plantsas an example of a eukaryotic organism not only in its authentic formbut also fused to a nuclear localisation signal.

First the recA gene was modified to remove most bacterial sequences fromits up- and downstream untranslated regions. In a second step, thecoding sequence was attached 5′ to the nuclear localisation sequence ofthe SV40 large T-antigen (Kalderon et al., 1984) yielding a fusionprotein (nt-RecA). To optimise nt-RecA translation, a leader sequencederived from the Rubisco SSU gene (Cashmore, 1983) encoding atranslation initiation codon was fused 5′ to the nt-RecA codingsequence. Both recA and nt-recA sequences were placed undertranscriptional control of the Cauliflower Mosaic Virus (CaMV) 35Spromoter and supplied 3′ with a eukaryotic polyadenylation signal (FIG.1). Finally, the genes were inserted into a binary vector suitable forAgrobacterium-mediated plant transformation using a sulfonamideresistance gene (Sul^(r)) to select transformed plants (FIG. 1). Inplasmid pS/recA the orientation of the recA transgene relative to theSul^(r) gene was such that both genes are transcribed in oppositedirections.

In plasmid pS/nt-recA the nt-recA and the Sul^(r) genes, however, aretranscribed in the same direction.

For biochemical characterisation, the nt-RecA fusion protein was alsoexpressed in E. coli. For this purpose plasmid pEV/nt-recA wasconstructed in which nt-recA was fused to an artificial ribosome bindingsite (FIG. 1).

The experimental details were as follows:

Modified recA genes were derived from plasmid pDR1453 (Sancar and Rupp,1979). The plasmid was digested with the restriction enzyme SacII, theends made blunt with DNA Polymerase I large fragment and theamino-terminal part of the recA gene subcloned as a SacII/EcoRI fragmentin plasmid pUC18, which had been cut with EcoRI and SmaI yieldingplasmid pRecA-1. The same plasmid was digested with HinfI, the endsrendered blunt and the carboxy-terminal part of the recA gene subclonedinto pUC19 (EcoRI/SmaI) as a HinfI/EcoRI fragment (pRecA-2). Theamino-terminal part was further modified. A BstXI/EcoRI and a TaqI/BstXIfragment obtained from pRecA-1 encoding the amino-terminal part of recAwithout its initiation codon and two complementary oligonucleotides(5′GGG GAC TCC TCC TAA GAA GAA GCG TAA GGT TAT GGC GAT3′ (SEQ ID NO: 1)and 5′CGA TCG CCA TAA CCT TAC GCT TCT TCT TAG GAG GAG TCC CC3′ (SEQ IDNO: 2)) encoding the missing codons as well as the SV40 nuclearlocalisation sequence were inserted into plasmid pUC18 which was hadbeen digested with EcoRI and SmaI, yielding plasmid pRecA-3. The DNAsequence of relevant junctions confirmed the expected structures of theconstructs.

For expression of nt-RecA in plants, the leader sequence and the codonsencoding the first 4 amino acids of the Rubisco SSU gene were fused tothe recA gene. Plasmid pSP64/TPNPTII (Wassmann et al., 1986) containsthe amino-terminal part of the SSU gene. This plasmid was digested withEcoRV and SalI and a SmaI/EcoRI fragment derived from pRecA-3 carryingthe amino-terminal portion of the recA gene and a EcoRI/SalI fragmentfrom pRecA-2 containing the carboxy-terminal portion inserted to it toyield pRecA-4. The complete nt-recA gene was excised from pRecA-4 bydigestion with HindIII and SalI. The HindIII ends were made blunt andthe fragment inserted into plasmid pDH51 (Pietrzak et al., 1986), whichhad been modified to contain an additional HindIII site upstream of theunique EcoRI site. This step fused the CaMV 35S promoter and thepolyadenylation signal, respectively, to the nt-recA gene. Forexpression of RecA in plants, the SmaI/EcoRI fragment from pRecA-3carrying the amino-terminal portion of the recA gene and the EcoRI/SalIfragment from pRecA-2 containing the carboxy-terminal portion wereinserted into the modified pDH51 plasmid via corresponding sites. Toobtain plasmid pEV/nt-recA for expression in E. coli, the BamHI/EcoRIfragment from pRecA-3 carrying the amino-terminal portion of the recAgene and the EcoRI/SalI fragment from pRecA-2 containing itscarboxy-terminal portion were inserted into plasmid pEVvfr1 (Crowl etal., 1985).

The binary vector carrying a Sul^(r) selectable marker gene wasconstructed as follows: Plasmid pJIT119 (Guerineau et al., 1990) wasdigested with HindIII, the ends filled in with DNA polymerase I largefragment, and the HindIII/SalI fragment carrying the Sul^(r) geneinserted into plasmid pDH51, which had been digested with SmaI and SalI.To obtain pS001, the Sul^(r) gene fused to the CaMV 35S promoter andpolyadenylation signal was exchanged for the methotrexate resistancegene in pM001 (Reiss et al., 1994) via the NcoI and SstI sites. The recAand nt-recA gene, respectively, was exised by digestion with HindIII andwas inserted into the unique HindIII site of plasmid pS001 leading toplasmids pS/recA and pS/nt-recA.

EXAMPLE 2 CHARACTERISATION OF NT-RECA

In order to determine whether fusion of the nuclear localisation signalhad any influence on the biochemical properties of RecA, a number ofreactions were tested: (I) In E. coli, RecA confers UV resistancedirectly via recombinational repair of replication blocks and indirectlyby mediating induction of SOS responses including increased expressionof RecA itself (Roberts et al., 1978). To test the function of nt-RecAplasmid pEV/nt-recA, in which the nt-recA gene was transcribed from thephage λP_(l) promoter, was introduced into the E.coli RecA- strain DH5α.

In particular, plasmid pEV/nt-recA was transformed into E. coli strainDH5α (supE44, ΔlacU169(Φ80lacZΔM15), hsdR17, recA1, endA1, gyrA96,thi-1, relA1, Gibco/BRL) which harboured a plasmid named 537 (Strebel etal., 1986) encoding a heat inducible lambda repressor gene. Cultureswere grown at 28° C. in LB medium supplemented with ampicillin (100μg/ml) and kanamycin (25 μ/ml). Expression was induced by a temperatureshift to 42° C. After an additional 2 hour growth and expression period,the cells were harvested by centrifugation and washed in 250 mM Tris/HClpH7.5, 25% (w/v) sucrose. High levels of expression, by thermalinactivation of the λcI857 repressor, turned out to be lethal. However,the cells were viable and UV-tolerant at 28 ° C., indicating thatnt-RecA was functional. Purification of nt-RecA was as described forRecA by Cox et al. (1981) with the modifications of Griffith and Shores(1985), yielding 20 mg nt-RecA from 2 g of cells. The protein was storedin 20 mM Tris/HCl pH7.5, 1 mM EDTA, 1 mM DTT buffer containing 20% (v/v)glycerol at −20° C. The protein concentration was determined accordingto Bradford (1976). The purity and identity of nt-RecA protein wasverified by SDS polyacrylamide gelelectrophoresis, Coomassie Bluestaining, and Western blotting using antibody ARM414, an antibodyproduced according to conventional procedures.

The preparation contained a single protein visible in the Coomassie Bluestain. This protein reacted with the anti-RecA antibody in the Westernblot. Using these criteria, the nt-RecA preparation was of equal puritywith a preparation of RecA protein which had been purified according tothe same protocol from a nalidixic-acid-induced E. coli cells harbouringplasmid pDR1453. (II) ATPase, ssDNA binding, and strand-exchangeactivities were analysed with highly purified nt-RecA protein producedby heat induction of a recAl E. coli strain carrying pEV/nt-recA.Purified nt-RecA preparations were shown to contain small amounts (lessthan 5%) of a protein of the molecular weight of authentic RecApresumably resulting from processing by an unspecific protease or fromtranslation starting at an internal initiation codon in nt-recA.

The ATPase activities of nt-RecA and authentic RecA, both purified fromE. coli cultures by the same procedure (Griffith and Shores, 1985), wereassayed in parallel in the presence and absence of ssDNA (Shibata etal., 1981). Essentially, ATPase activity was determined as described byShibata et al. (1981) except that [³H]ATP was substituted by [¹⁴C]ATP(Amersham, specific activity 5×10¹² Ci/Mol). The basal ATPase activityof authentic RecA was found to be low but to be stimulated 20-fold bythe addition of ssDNA, as expected (see Roca and Cox, 1990). Incontrast, the basal ATPase activity of nt-RecA was found to be muchhigher and stimulated proportionally less by the addition of ssDNA (FIG.2). (III) Binding of purified nt-RecA to ssDNA was assayed using gelretardation assays. A fixed quantity of ssDNA was incubated withincreasing amounts of purified RecA and nt-RecA protein in the presenceof ySATP to prevent dissociation of the complexes. In particular,binding of nt-RecA and RecA to ssDNA was determined in a total volume of20 μl 25 mM Tris/acetate, 4 mM MgCl₂, 1 mM DTT, 20 μM nucleotide ssDNAfrom a derivative of phage M13mp18, described by Wada et al. (1994), and2 m μ γSATP. Different quantities of protein (0, 20, 50, and 100 pMol)were incubated for 30 min at 37° C. with this mixture. Fordeproteinisation, SDS and EDTA were added to final concentrations of 1%(w/v) and 10 mM, respectively (Riddles and Lehmann, 1985). Nodifferences in binding kinetics were observed (FIG. 3). (IV) RecA andnt-RecA proteins promoted strand-exchange between linear dsDNA andcircular ssDNA (Menetski et al., 1990) with the same kinetics (FIG. 4).The strand-exchange reaction was performed exactly as described byMenetski et al. (1990). Closed circular ssDNA and Bg1I linearised dsDNAprepared from a derivative of phage M13mp18 (Wada et al., 1994) wereused as substrates. These tests therefore indicated that nt-RecAexhibited the activities expected of a RecA protein.

EXAMPLE 3 CHARACTERISATION OF RECA AND NT-RECA TRANSGENIC PLANTS

Agrobacteria harbouring binary vectors carrying the recA and nt-recAtransgenes were used to infect tobacco leaf disks: Plasmids pS/recA andpS/nt-recA were transferred to Agrobacterium tumefaciens strainGV3101/pMP90RK (Koncz and Schell, 1986) via electroporation, and theresulting strains (plants transgenic for recA were designated G64 andthe nt-recA transgenic plants G63) used to inoculate leaf disks madefrom sterile tobacco SR1 plants according to published procedures (Konczand Schell, 1986). Transformed shoots were selected on sulfadiazine (100mg/l). Plants were regenerated and tested for rooting on sulfadiazine(100 mg/l). Transgenic plants were grown to maturity in the green houseand seeds harvested. The inheritance of the transgenes was tested bygermination of seeds in the presence of sulfadiazine on the same media.SR1hph2 plants were grown from seedlings which were selected onhygromycin (15 mg/l) under sterile conditions. G63 and G64 plants werecrossed to SR1hph2 plants in the green house. Siblings harbouring therecA and hph2 transgenes were selected by growth of seedlings in thepresence of both sulfadiazine (100 mg/l) and hygromycin (15 mg/l) understerile conditions. Plants were grown to maturity without furtherselection. Individual transgenic plants were numbered consecutively.Shoots which rooted on selective media containing sulfonamide wereconsidered to be resistant and were selected for further analysis.

The presence of recA transgenes was confirmed by Southern blots. ForSouthern hybridisations, total DNA prepared from leaves (Murray andThompson, 1980) was restricted with EcoRI, and the fragments separatedby agarose gel electrophoresis and blotted to a Nylon membrane(Zetaprobe, Biorad). The membrane was hybridised according to themanufacturers guidelines, to radioactively labeled probes prepared asdescribed by Feinberg and Vogelstein (1984). Fragments encoding recAsequences were detected using a fragment from pRecA-4 which covered theentire gene. The copy number of inserts was determined using probesspecific for border fragments which were derived from the Sulr. and recAgenes. It was found that 11 of 12 sulfonamide resistant G64 plantscarried an intact recA transgene. Plants with single copy inserts wereselected and shown to transmit the sulfonamide resistance marker totheir progeny as a single Mendelian trait. In contrast, only three of 36G63 plants were found to carry an intact nt-recA gene. In one of them,G63/19, the right border of the T-DNA was deleted. The deletion resultedin a fusion of the 35S promoter sequences controlling nt-recA expressionto plant genomic sequences. Other transgenic plant lines harbouredeither no recA sequences or had rearranged nt-recA genes. The intactnt-recA transgenes in the three independent transgenic plants were shownto be present in single copy and to be inherited as a single Mendeliantrait.

Expression of the recA and nt-recA genes was monitored using Westernblots of protein extracts of leaves probed with a RecA-specificmonoclonal mouse antibody (ARM414, Ikeda et al.,1990). First, proteinswere extracted from leaves of plants grown in sterile culture. Leaveswere ground with Laemmli sample buffer (Laemmli, 1970) (withoutbromphenol blue) plus sea sand, using a glass rod in an Eppendorf tube.After heat denaturation of the samples for 15 min at 95° C., the extractwas cleared by centrifugation and the supernatant used for furtheranalysis. The protein concentration was determined (Bradford, 1976) and50 μg used for electrophoresis on polyacrylamide SDS gels according toLaemmli (1970). Proteins were transferred to nitrocellulose membranes(Schleicher and Schuell, pore size 0.45 μm) as described (Towbin, 1979).RecA protein was detected using the monoclonal anti-RecA antibody ARM414(Ikeda et al., 1990) in a 1:200 dilution in TBST buffer after blockingof non-specific protein binding by 5% non-fat dry milk. A secondantibody (goat anti-mouse, Promega) coupled to alkaline phosphatase andNBT/BCIP (Promega) staining or a second anti-mouse antibody coupled tohorse radish peroxidase and chemiluminescence (ECL, Amersham) was usedto develop the blot. All plants with an intact recA or nt-recA gene werethus shown to express RecA protein. Expression in G64 plants (recA) wassimilar in most transgenics and amounted to about 0.1% of total plantprotein, as judged from a comparison of staining intensities with thoseof a dilution series of purified RecA protein. The expression level ofnt-recA ranged from 0.01% (G63/17) to 0.1% (G63/19) of total protein.Both RecA and nt-RecA proteins appeared stable in plant cells and showedthe expected molecular weights (FIG. 5). In nt-recA transgenic plants,small amounts of additional proteins were detected with the antibody.Since these proteins were of lower molecular weight, they presumablywere degradation products of the actual nt-RecA protein.

The localisation of the RecA and nt-RecA proteins in the cell wasstudied by indirect immunbfluorescence. Plants with comparableexpression levels were selected (G64/2 and G63/19) and protoplasts orroot squashes were prepared. The preparation of protoplasts was done,according to the method of Negrutiu (1987), for immuno-histochemicallocalisation of RecA and nt-RecA protein. Proteins were fixed with 5%formaldehyde (10⁵ protoplasts in 2 ml K3, 0.4M sucrose) at roomtemperature. Formaldeyde was removed by washing in W5 and chlorophyllextracted with methanol. Nonspecific binding was blocked by anincubation of the protoplast preparation in 5% BSA in TBST for 1 hour.The protoplasts were collected by centrifugation and incubated withanti-RecA antibody ARM414 (1:50) in TBST. After extensive washing withTBST buffer containing 5% BSA, the protoplasts were incubated withFITC-labelled anti-mouse antibody (Promega, 1:1000). Unbound antibodywas removed by washing with TBST 5% BSA. Nuclei were stained with DAPI(2 μg/ml) in the same buffer. The preparation was examined usingfluorescence microscopy (Zeiss Axiophot).

RecA and nt-RecA proteins were localised in whole tissue from youngplants after fixation of leaves or roots in methanol:acetic acid (3:1)for 1 hour at room temperature. Tissue was equilibrated with K3 mediumand incubated over night with 0.9% cellulase in the same medium. Afterincubation in acetic acid for 5 min, the tissue was transferred to aslide and squashed. Staining with anti-RecA antibody was as describedabove for protoplasts.

In G63/19 plants, FITC-fluorescence was found almost exclusively in thenucleus (FIG. 6). Some staining was visible also in the chloroplasts. Incontrast, in G64/2 cells the nuclei were not particularly stained.However, there seemed to be a weak preference for association with thenucleus and concentration in the region around the nucleus (FIG. 6).Only background staining was observed in non-transgenic SR1 tobaccoplants. It can be concluded that the SV40 nuclear localisation sequencein nt-RecA leads to efficient accumulation of this protein in thenucleus of the plant cell.

EXAMPLE 4 EXPRESSION OF RECA AND NT-RECA LEADS TO INCREASED RESISTANCETO MITOMYCIN C

To analyse the effect of mitomycin C on plant growth, a quantitative andreproducible assay was used. The system described by Lebel et al. (1993)which allows to follow the fate of single plant cells was developedfurther to obtain greater sensitivity to mitomycin C and a monotonicsurvival-dose-response curve.

In this system protoplasts were prepared from sterile-grown tobacco SR1plants and parallel preparations treated with various concentrations ofmitomycin C. Subsequently the protoplasts were cultivated in a bead-typeculture in the presence of mitomycin C. Untreated protoplasts activelydivided and formed microcalli within a period of 4 to 8 weeks. Theexperimental details were as follows:

Protoplasts were prepared from leaves of axenically grown plants asdescribed by Negrutiu (1987), with some modifications. Cut leaves (3 g)were digested in 50 ml K3, 0.4 M sucrose, 1 mg/l NAA, 0.2 mg/l kinetin,0.6% cellulase Onozuka R10 (Serva), 0.3% Macerozyme R10 (Serva) in 145mmpetri dishes at 22° C., in the dark, for 16 hours. Protoplasts werepurified by filtration through steel sieves (250 μm and 100 μm meshwidth) and washed once in W5 medium. Protoplasts were suspended in 1 mlof MaMg buffer (0.5 M mannitol, 15 mM MgCl₂, 0.1% MES, pH5.7), countedunder a light microscope and diluted to a final concentration of 10⁶cells/ml with K3, 0.4 M sucrose. A 1 ml aliquot of protoplast solutionwas diluted with 9 ml K3, 0.4 M sucrose medium and mitomycin C addedfrom a stock solution, to the final concentrations indicated in FIG. 7.After incubation for 2 days in the dark at 22° C., the protoplasts werecultivated using the bead-type technique of Shillito et al. (1983) withsome modifications. Protoplasts were embedded by dilution with an equalvolume of media containing 0.8% low-melting-point agarose (FMC), as agelling agent, and grown on a solid support carrier system (paper filterdiscs) in 20 ml liquid media. Cultures were grown for approximately 4weeks with weekly changes of media. Survival was scored when themicrocalli reached sizes of 2 to 4 mm. Plants were regenerated fromrepresentative samples. These plants showed no obvious growthabnormalities.

In a typical control experiment, 10% to 20% of the protoplasts platedgrew to microcalli. Increasing concentrations of mitomycin Cprogressively inhibited the formation of microcalli (FIG. 7). No growth(less than 10⁻³% of control values) was observed at concentrations of 40μ/ml and above. The survival curve showed a low-dose shoulder suggestingthe presence of repair mechanisms leading to resistance to mitomycin Cfollowed by a semi-logarithmic region at high doses of mitomycin Ccausing damage which can no longer be repaired by the endogenous repairmechanisms.

Protoplasts of a plant homozygous for the recA transgene (G64/2) wereslightly but significantly more resistant to the toxic effect ofmitomycin C than control cells. At concentrations of 40 μg/ml mitomycinC more than 0.1% of the cells survived and grew to microcalli (FIG. 7).However, no recA transgenic cells (less than 10⁻³% of control values)grew at mitomycin C concentrations of 50 μg/ml and above. In contrast,more than 0.1% nt-recA transgenic protoplasts (homozygous G63/19) wereable to grow and regenerate at concentrations of up to 60 μ/ml mitomycinC, the highest concentration tested (FIG. 7).

EXAMPLE 5 INTRACHROMOSOMAL RECOMBINATION OF A CHROMOSOMAL MARKER ISSTIMULATED BY RECA AND NT-RECA

Plants usually contain large amounts of repetitive DNA sequences.Recombination within these sequences apparently played a role in genomeevolution (for review see: Flavell, 1982). To study the process ofintrachromosomal recombination in plants, Peterhans et al. (1990) havedeveloped a transgenic system. A pair of deletion derivatives of theselectable marker gene neomycin phosphotransferase (nptII, Beck et al.,1982) were stably integrated into the tobacco genome. The deletionsremoved portions of either the 5′ or the 3′ end of the gene rendering itnon-functional. The segments in line SR1hph2 (Peterhans et al., 1990)were oriented as direct repeats with a 352-bp homologous overlap,interrupted by a functional hygromycin phosphotransferase gene (Van denElzen et al., 1985). In this line, the basic module was present in threetightly linked copies in the genome. Intrachromosomal recombinationevents which lead to restoration of a functional nptII gene can easilybe detected by selection of kanamycin resistant cells in tissue culture.

To study the influence of RecA and nt-RecA expression onintrachromosomal recombination, line SR1hph2 containing the defectivenptII genes was crossed respectively to the homozygous lines G64/2 andG63/19. Progeny plants carrying the recA respectively nt-recA genes aswell as the defective nptII genes were selected by germinating seeds onhygromycin and sulfonamide. Plants resistant to both antibiotics weregrown under sterile culture conditions without further selection andleaf mesophyll protoplasts were prepared as described in Example 4. Todetermine the number of intrachromosomal recombination events, cultureswere grown for 6 to 8 weeks in the presence of 100 μ/ml kanamycin afterembedding. To determine the regeneration frequency, protoplasts weregrown under identical conditions without kanamycin. From representativesamples of microcalli, plants were regenerated to verify resistance tokanamycin. Protoplasts were plated and cultured until microcalliappeared. The number of protoplasts forming microcalli in the absence ofselection (regeneration frequency) was determined for each batch ofprotoplasts and found to be about 20% to 30% for all protoplastpreparations. The number of protoplasts regenerating in the presence ofkanamycin was determined. The frequency of intrachromosomalrecombination was calculated from the number of microcalli which grew onkanamycin versus the total number of calli appearing on non-selectivemedia. 20 kanamycin-resistant microcalli were selected at random forregeneration into plants. All regenerated plants formed roots onkanamycin-containing medium, confirming that calli which grew in thepresence of kanamycin were indeed resistant to the antibiotic.

The frequency of intrachromosomal recombination was found to be1.04×10⁻⁵ in the control line SR1hph2. In contrast, the frequencies inG64/2 X SR1hph2 and G63/19 X SR1hph2 were found to be 5.37×10⁻⁵ and10.3×10⁻⁵, respectively (FIG. 8). These data show that the RecA protein,especially if targeted to the nucleus, is able to interact with theplant chromosome and the host recombination machinery and to markedlyincrease the level of intrachromosomal somatic recombination.

EXAMPLE 6 ENHANCEMENT OF GENE TARGETING FREQUENCIES IN PLANTS BY ECTOPICNT-RECA EXPRESSION

A chimeric target locus was generated which consists of the seedspecific high molecular weight glutenin (HMW) promoter (Colot et al.,1987) and additional sequences consisting of pBR322 and a selectablemarker conferring methotrexate resistance in plants. To obtain thisconstruct, the HMW promoter was cloned as an EcoRI/BamHI fragment intothe binary vector pMN001 (Reiss et al., 1994). The resulting plasmid wastransferred via electroporation to Agrobacterium (GV3101pMP90RK, Konczand Schell, 1986) and the resulting strains were used to generatetransgenic tobacco SR1 plants by leaf disk infection according topublished methods (Marton et al., 1982; De Block et al., 1984; Marton,1984; Horsch et al., 1985). These plants were characterized by Southernblotting and one line which contained the chimeric target locus insingle copy designated B18/4. This line expressed NPT II in seeds, butno activity was detected in leaves using a sensitive enzymatic assay(Reiss et al., 1984). When callus was induced on leaf disks from theseplants, green living material readily developed on methotrexate, but nocallus formed on kanamycin. These plants were crossed to G63/19 SR1plants expressing the nt-RecA protein (Reiss et al., 1996).

A repair construct was made which would lead to constitutive expressionof NPT II upon homologous recombination with the B18/4 target. Thisconstruct was identical to the one used to generate B18/4, but containeda promoter expressed in all tissues, the CaMV 35S promoter, insertedinto the BamHI site between the HMW promoter and the npt II gene and thenpt II gene was the non-functional variant D42 which contained acarboxyterminal deletion shown to yield no active protein in E. coli(Beck et al., 1982). This plasmid was introduced into Agrobacterium(GV3101pMP90RK) as described above to yield strain G125.

To analyze gene targeting, siblings obtained from G63/19 plants crossedto B18/4 were selected on methotrexate and sulfonamide to select for thepresence of both sets of transgenes. Leaf disks were prepared andtransformed with Agrobacterium strain G125 which harboured the repairconstruct. In control transformations, 500 calli were obtained with G125on a total of 58 SR1 leaf disks after selection on methotrexate. Thisdemonstrated that G125 was fully functional. In total, 189 leaf disksfrom B18/4×G63/19 were infected with G125 and 21 kanamycin resistancecalli obtained after selection on kanamycin.

To determine whether the resistant calli derived from potential genetargeting events, total genomic DNA was prepared from 9 of them. The DNAwas amplified by PCR with a primer pair specific for an intact npt IIgene under 35S promoter control (Primer 1 homologous to the −90 regionof the 35S promoter: 5′GTG GAT TGA TGT GAT ATC TCC3′ (SEQ ID NO: 3);Primer 2 homologous to sequences of npt II deleted in D42: 5′CCG CTC AGAAGA ACT CGT CA3′ (SEQ ID NO: 4)). A fragment of the size predicted foramplification of the restored nptII gene with this primer pair wasobtained in six calli. No amplification product was obtained with DNAfrom the parental B18/4 line and the residual 3 calli. These resultsindicate the presence of an intact npt II gene under 35S promotercontrol in 6 of the 9 calli investigated. The npt II gene in the 3kanamycin resistant, but PCR negative calli most likely was activated bysomaclonal variation, not by gene targeting.

The frequency of restoration of an intact npt II gene expressed in leaftissue by transformation with G125 can be calculated therefore asfollows:

The transformation frequency in the control experiment was 500transformants per 58 leaf disks or 8.6/leaf disk. Thus, it was to beexpected that in a total of 189 B18/4×G63/19 leaf disks transformed,approximately 189×8,6=1625 transformants were generated withoutselection on kanamycin. A number of 21 kanamycin resistant calli wasdetected. Therefore, these calli appeared with a frequency of 21/1625which approximately equals 1.3%. In a representative sample of 9, 6contained a restored npt II gene. Therefore the frequency of targetingin this experiment was 0.87%.

Transgenic target loci similar to the system described here were usedpreviously. The targeting frequencies observed with those target lociusing Agrobacterium-mediated transformation were in the order of 10⁻⁴(Offringa et al., 1990). Although these experiments might not bedirectly comparable, the large increase in frequencies observed in ourexperiments indicate a stimulatory role of RecA expression.

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8 1 39 DNA Artificial Sequence Description of Artificial Sequenceamino-terminal part of recA without its initiation codon and twocomplementary oligonucleotides 1 ggggactcct cctaagaaga agcgtaaggttatggcgat 39 2 41 DNA Artificial Sequence Description of ArtificialSequence portion of recA and SV40 nuclear localization sequence 2cgatcgccat aaccttacgc ttcttcttag gaggagtccc c 41 3 21 DNA ArtificialSequence Description of Artificial Sequence primer homologous to the -90region of the 35S promoter 3 gtggattagt gtgatatctc c 21 4 20 DNAArtificial Sequence Description of Artificial Sequence primer homologousto the sequence of the nptII gene 4 ccgctcagaa gaactcgtca 20 5 45 DNAArtificial Sequence Description of Artificial Sequence nuclear targetingsequence 5 atggcttcta tgatggggac tcctcctaag aagaagcgta aggtt 45 6 15 PRTArtificial Sequence Description of Artificial Sequence nuclear targetingsequence 6 Met Ala Ser Met Met Gly Thr Pro Pro Lys Lys Lys Arg Lys Val 15 10 15 7 51 DNA Artificial Sequence Description of Artificial Sequenceartificial ribosome binding site 7 atgaatgaat tcggatcccc ggggactcctcctaagaaga agcgtaaggt t 51 8 17 PRT Artificial Sequence Description ofArtificial Sequence artificial ribosome binding site 8 Met Asn Glu PheGly Ser Pro Gly Thr Pro Pro Lys Lys Lys Arg Lys 1 5 10 15 Val

What is claimed is:
 1. A method for the production of a transgenic plantor a transgenic plant cell with enhanced recombination, comprising (1)insertion of at least a first and second DNA sequence into the genome ofsaid plant or plant cell, said first DNA comprising (a) the sequence ofat least one gene of interest, and (b) a sequence which encodes aselection marker expressible in said plant or plant cell, and saidsecond DNA comprising a sequence that encodes a RecA operatively linkedto a DNA sequence encoding a nuclear targeting sequence for promotinghomologous recombination, and (2) selection of transgenic plants orplant cells which have taken up said DNA, and (3) culturing of saidtransgenic plant or transgenic plant cells in a suitable culture medium.2. The method according to claim 1, wherein said transgenic plant ortransgenic plant cell is Nicotiana tabacum or Arabidopsis thaliana.
 3. Amethod for the production of a transgenic plant or a transgenic plantcell with enhanced recombination, comprising (1) insertion of DNA intothe genome of a plant or plant cell, said DNA comprising (a) thesequence of at least one gene of interest, and (b) a sequence whichencodes a selection marker expressible in said plant or plant cell, and(c) a nucleic acid molecule encoding the amino acid sequence of the E.coli RecA protein, wherein said nucleic acid molecule is operativelylinked to a DNA sequence encoding a nuclear targeting sequence, (2)selection of desired transgenic plants or transgenic plant cells whichhave taken up said DNA, and (3) culturing of said desired transgenicplants or transgenic plant cells in a suitable culture medium.
 4. Themethod according to claim 1 or 3, wherein said selection marker isHyg^(R), Km^(R), PPT^(R), Mtx^(R) or Sul^(R).
 5. A transgenic plant ortransgenic plant cell prepared by the method of claim
 1. 6. A vectorwhich comprises a DNA sequence encoding a nuclear targeting sequenceoperatively linked to a sequence that encodes a RecA and furthercomprising a DNA sequence encoding a selectable marker.
 7. A vectorwhich comprises a DNA sequence encoding a T SV40 nuclear targetingsequence operatively linked to a sequence that encodes a RecA andfurther comprising a DNA sequence encoding a selection marker and theDNA sequence of at least one gene of interest.
 8. The method of claim 1or 3, wherein said nuclear targeting sequence is that of T SV40.
 9. Themethod according to claim 1, wherein said nucleic acid molecule encodesE. coli RecA.
 10. The method according to claim 1 or 3, wherein saidinsertion is mediated via a member selected from the group consisting ofPEG transformation, Agrobacterium transformation, electroporation,particle bombardment, liposome fusion, in planta transformation, calciumphosphate precipitation and virus infection.
 11. A method for theproduction of a transgenic plant or a transgenic plant cell withenhanced recombination, comprising (1) insertion of DNA into the genomeof a plant or plant cell, said DNA comprising (a) the sequence of atleast one gene of interest, and (b) a sequence which encodes a selectionmarker expressible in said plant or plant cell, and (c) a nucleic acidmolecule encoding the amino acid sequence of the E. coli RecA protein,and wherein said nucleic acid molecule of (c) is operatively linked to aDNA sequence encoding the T SV40 nuclear targeting sequence, (2)selection of desired transgenic plants or transgenic plant cells whichhave taken up said DNA, and (3) culturing of said desired transgenicplants or transgenic cells in a suitable culture medium.
 12. The methodaccording to claim 11, wherein said insertion is mediated via a memberselected from the group consisting of PEG transformation, Agrobactenumtransformation, electroporation, particle bombardment, liposome fusion,in planta transformation, calcium phosphate precipitation and virusinfection.
 13. A vector which comprises a DNA sequence encoding a T SV40nuclear targeting sequence operatively linked to a a nucleic acidsequence encoding the amino acid sequence of the E. coli RecA protein,wherein said vector further comprises a promoter that functions inplants, said promoter operably linked to said DNA sequence.
 14. Thevector according to claim 13, further comprising a DNA sequence encodinga selection marker.
 15. The vector according to claim 13, furthercomprising at least one gene of interest.
 16. A vector which comprises aDNA sequence encoding a T SV40 nuclear targeting sequence operativelylinked to a sequence that encodes RecA that mediates strand-exchange andfurther comprising a DNA sequence encoding a selection marker and theDNA sequence of at least one gene of interest.
 17. The vector accordingto claim 6, wherein said nuclear targeting sequence is the T SV40nuclear targeting sequence or the RecA is the E. coli RecA protein. 18.The vector according to claim 7, wherein the RecA is the E. coli RecAprotein.
 19. The vector according to claim 17 or 18, wherein said vectoris pS/nt-RecA or pEV/nt-RecA.
 20. The method according to claim 9,wherein said nuclear targeting sequence is that of T SV40.